Tumor necrosis factor (TNF; previously referred to as tumor necrosis factor-α) is a proinflammatory cytokine that plays a major role in the pathogenesis of rheumatoid arthritis and associated inflammatory diseases, such as ankylosing spondylitis, juvenile rheumatoid arthritis, and psoriatic arthritis. The human proform, transmembrane-bound TNF (tmTNF), is a 26-kDa homotrimer comprising three non-covalently associated monomers, each monomer having N-terminal sequence imbedded in the cell membrane. Each monomer of tmTNF has a 233 amino acid sequence (UniProtKB/Swiss-Prot entry Accession No. P01375). Soluble TNF (sTNF) is a homotrimer formed by enzymatic cleavage from its pro-form tmTNF. Each monomer of the sTNF trimer has a 157 amino acid sequence (SEQ ID NO: 1), which is the same sequence as aa77 to 233 of the published Acc No. P01374.
Both forms of active TNF (tmTNF and sTNF) exist as homotrimers10-13 and engage trimeric receptors that recognize receptor-binding sites in the grooves between the TNF monomers in assembled homotrimers. The grooves between the monomers comprise amino acid sequence from two contiguous monomers14,15. The receptor binding regions of both forms of TNF are identical.
Trimer integrity is essential for biological function. For tmTNF, trimeric structure is established intracellularly before tmTNF insertion into the cell membrane 16 and is maintained in tmTNF by the anchoring of the protein stems passing through the membrane plus further lipid anchoring by palmitoylated amino acid side chains at the membrane boundary17. In contrast, sTNF active trimers dissociate freely into inactive monomers SEQ ID NO: 1 and dimers that reform as active sTNF homotrimers in a steady-state equilibrium between the three forms18.
Anti-TNF biologics have provided a major advance in the management of the above-noted inflammatory diseases with anti-TNF monoclonal antibodies REMICADE (Infliximab; Janssen Biotech, Inc.) and HUMIRA (Adalimumab, Abbott Laboratories), and a chimeric solubilized TNF receptor fused to Fc, i.e., ENBREL (Entanercept, Biogen, Inc) being widely used1,2. This therapeutic and marketing success is marred by the rare but statistically significant occurrence of serious infections and malignancies3,4, likely related to concomitant blockade of tmTNF5,6 function impairing immune defenses. These adverse occurrences have included the development of tuberculosis, systemic fungal infection and other intracellular infections due principally to intracellular pathogens such as Mycobacterium tuberculosis, Listeria monocytogenes and Histoplasma capsulatum, and certain forms of cancer. These results were unsurprising since these agents block pro-inflammatory sTNF but also block tmTNF, which is essential for juxtacrine cellular control of such intracellular infections and malignancies3,7,9.
Because the receptor binding regions of both forms of TNF are identical, there has been little hope for the development of new monoclonal antibodies selectively blocking receptor engagements of one form versus the other. Antibodies to short sequences of TNF have not lead to useful therapeutics. For example, in 1987, Socher et al.26 in exploring antibodies to full or partial synthetic sequences of TNF, observed a high polyclonal antibody response to the TNF fragment 1-15 that appeared to block bioactivity and receptor binding of TNF. However, this 16-year old observation has not lead to the development of additional therapeutic reagents, likely because the TNF receptor is a discontinuous surface region not associated with TNF amino acids 1-15. Subsequent researchers in 2001 coupled TNF amino acids 4-23 conjugated to papillomavirus-like particles, and observed an induction of polyclonal antibodies, and an attenuation of experimental arthritis27. Other researchers in 2007 used the same fragment TNF aa4-23 coupled to a virus-like particle-based composition and induced antibodies that attenuated experimental arthritis. No suppression of resistance to infection occurred, in contrast with full length TNF immunization28. Because these TNF fragments were not directed to receptor binding regions of TNF, these publications displayed no further teachings or suggestion of therapeutic use of the resulting polyclonal antibodies; and further research has not been published since that date.
One more recent attempt to selectively suppress the pro-inflammatory activity of sTNF while preserving tmTNF function required for innate immunity involved the design of synthetic dominant-negative TNF monomer variants that formed trimers that were inactive19. These were shown to attenuate experimental arthritis without suppressing innate immunity to infection20, emphasizing the major role of sTNF in pathogenesis of arthritis. Another approach has been the search for small-molecule drugs that interact with the inter-monomer contact regions. One molecule, SP304, bound such a contact region with μM affinity to effect trimer disruption in vitro21,22.
Despite the plethora of literature in the field of anti-TNF treatment for a variety of inflammatory disorders, there remains a need in the art for new and useful compositions and methods for generating therapeutic or prophylactic immunogenic compositions for these diseases which do not result in adverse side effects due to suppression of cellular immunity.